No abstract

The sensitivity of Venturia inaequalis field isolates to inhibitors of the cytochrome bc1 complex at the Qo site (QoIs) was characterised at the molecular, biochemical and physiological level, and compared to other respiration inhibitors. Comparison of a sensitive and a QoI-resistant isolate revealed very high resistance factors both in mycelium growth and spore germination assays. Cross-resistance was observed among QoIs such as trifloxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. In the mycelium growth assay, antimycin A, an inhibitor of the cytochrome bc1 complex at the Qi site, was less active against the QoI-resistant than against the sensitive isolate. The mixture of QoIs with salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase, exerted synergistic effects in the spore germination but not in the mycelium growth assay. Thus, the cytochrome and the alternative respiration pathways are assumed to play different roles, depending on the developmental stage of the fungus. Induction of alternative oxidase (AOX) by trifloxystrobin was observed in mycelium cells at the molecular level for the sensitive but not the resistant isolate. Following QoI treatment, respiration parameters such as oxygen consumption, ATP level, membrane potential and succinate dehydrogenase activity were only slightly reduced in Qo-resistant mycelium cells, and remained at much higher levels than in sensitive cells. In contrast, no difference was observed between sensitive and resistant isolates when NADH consumption was measured. Comparison of the cytochrome b (cyt b) gene of the sensitive and resistant isolates did not reveal any point mutations as is known to occur in resistant isolates of other plant pathogens. It is assumed that QoI resistance in V inaequalis may be based on a compensation of the energy deficiency following QoI application upstream of the NADH dehydrogenase of the respiratory chain.

Field isolates of Mycosphaerella ®jiensis, causing black Sigatoka of banana, were characterised for their sensitivity to different inhibitors of the cytochrome bc 1 enzyme complex (Qo respiration inhibitors, strobilurin fungicides), using physiological, biochemical and molecular genetic methods. Strobilurin-resistant isolates exhibited very high resistance factors both in mycelial growth inhibition and NADH consumption assays. Cross-resistance was observed among all Qo inhibitors, including tri¯oxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. However, the Qi and the cytochrome aa 3 inhibitors, antimycin A and potassium cyanide, respectively, were not crossresistant to Qo inhibitors. In sensitive but not in resistant isolates, mixtures of Qo inhibitors and SHAM, an inhibitor of the alternative oxidase (AOX), were more active than the components alone, indicating that the alternative pathway is essential in metabolism, but not causal for resistance. In the cell-free NADH-consumption assay, the Qo inhibitors affected the sensitive but not the resistant isolates, suggesting that AOX was not active in sub-mitochondrial particles. In whole cells, however, the AOX has a basic expression level and is probably not inducible by tri¯oxystrobin. Sequencing of the cytochrome b gene of sensitive and resistant M ®jiensis isolates revealed a difference in the nucleotide sequence leading to a single amino acid change from glycine to alanine at position 143 in the resistant isolate. This change is known to occur also in the naturally tolerant basidiomycete Mycena galopoda. It is suggested that the ®eld isolates of M ®jiensis can acquire resistance to Qo inhibitors due to a target site alteration with a single base pair change. Resistant isolates do not seem to contain a mixture of mutated and non-mutated DNA, indicating a complete selection of resistant mitochondria and a maternally donated mode of resistance.